Exonuclease Iii-Assisted Crispr/Cas12a Electrochemiluminescence Biosensor for Sub-Femtomolar Mercury Ions Determination

CRISPR/Cas12a, due to its prominently tunable cleavage ability, has been attracted large attention in biosensing field such as the detection of nucleic acids, proteins, small molecules and so on. Nevertheless, its application on heavy metal ions determination has been rarely reported. Herein, we developed a ECL biosensor integrating Exo III and CRISPR/Cas12a to achieve ultrasensitive determination of mercury ions. The Hg 2+ was first converted into an amplified activator DNA via Exo III-mediated enzymatical degradation coordination to release Hg 2+ for the next cycle. Simultaneously, the countless of activator DNA was produced and hybridized with crRNA in the Cas12a-crRNA duplex and activate the collateral cleavage capability of Cas12a to indiscriminately cleave the ferrocene-labeled single-stranded DNA (Fc-ssDNA) on the electrode. Whereafter, the Fc was released from the surface of electrode to attenuate quenching effect for the ECL emission of gold nanoparticles-coated nitrogen-doped carbon dots (Au@CNDs). The concentration of Hg 2+ was directly quantitative through the ECL signal. By virtue of the Exo III-mediated enzymatical degradation and the collateral cleavage capability of Cas12a, a detection limit at sub-femtomolar level was realized. The biosensor extended the application of CRISPR/Cas systems and provided a promising strategy for other toxic metal ions determination.